K562 human erythroleukemia cells constitutively express epsilon and gamma, but not beta-globin genes. The different expression of globin genes observed in intact K562 cells could be simulated in vitro as K562 nuclear extract (NE) actively transcribes the epsilon and gamma-globin gene DNA templates, but not the beta-globin gene. We have used the K562 in vitro transcription system to examine a transcriptional control element between - 392 and -177bp 5' of the canonical cap site for the epsilon-globin gene which has been reported to function as a silencer. We find that K562 NE cannot actively synthesize RNA in vitro from the epsilon-globin gene DNA deletion templates which contain the entire or a part of the silencer sequence but no positive regulatory region. Separating the K562 NE by ion exchange chromatography, we isolated a transcriptionally active fraction (F175) for globin genes and a fraction (F50) which contains the trans- acting factors associated with the silencer activity. The F175 was transcriptionally active for all tested globin genes including the epsilon- globin gene containing the silencer sequence. In contrast, F50 showed a strong dose dependent inhibitory effect on epsilon-globin gene transcription directed by either unfractionated K562 NE or F175. This suppression by F50 was not observed on transcription of adenovirus 2 major late promoter. In electrophoretic mobility shift assays using the epsilon- globin gene silencer region as probe, F50 and F175 exhibited different DNA binding protein patterns: a specific protein band in F50 appears to be associated with the silencer activity. These studies suggest that this protein may be specifically responsible for the activity of the silencer element of the epsilon-globin gene and that the expression and silencing of the epsilon-globin gene during development may be modulated by the interactions of this protein with the cis-acting DNA silencer.